1. Field of the Invention
This invention relates to immunochemicalassaying. Immunochemicalassays are proving of immense value in medicine and biology for assaying liquid samples, especially, for example, body fluid samples such as blood or urine, because of the sensitivity and specificity of such assays. The present invention is concerned with assaying for phenobarbital, phenyl methyl barbituric acid and mephobarbital and related hydrantoin compounds, including diphenyl hydantoin. Accurate assay of these substances is of the utmost value in medical diagnosis and control of drug abuse.
In immunoassaying procedures, for a given target compound, a synthetic antigen is generally first prepared. Heretofore, this has usually been accomplished by coupling the target compound, through a coupling group to a carrier which confers antigenicity to the entire compound. The compound coupled to the carrier is usually known as a hapten and, when coupled, it functions as an antigenic determinant so that the antibodies produced will bind with the hapten. Thus, the antibodies produced should have a distinct and unique character, such that they will bind with only a specific compound or class of compounds. The objective in devising the synthetic hapten-carrier conjugate is to provide a compound which will generate antibodies that are specific to the target compound.
Antibodies are prepared by injecting the synthetic hapten-carrier conjugate into mammals and recovering blood serum from the mammals after they have had time to generate antibodies. Typical mammals are rabbits and goats.
The principal problem is usually that of synthesizing antigens that are capable of producing sufficiently specific antibodies. Biological fluids such as blood and urine frequently contain very closely related compounds and it is common for antibodies to be unable to distinguish the target compound from close relatives, or sometimes even from distant ones. The antibody is then considered to be a poor one and is said to have low specificity and high cross-reactivity.
The assay itself is commonly a competitive binding assay. In a useful embodiment of such an assay, the target compound, which is not necessarily extracted, is allowed to compete with known quantities of a labeled standard to bind with a known quantity of specific antibody. From measurement of the proportion of the labeling in the standard-antibody complex that results, the amount of target compound present can be calculated. Radioactive labeling is particularly convenient. Fluorescence perturbation and electron spin resonance have been used in the art. Normally it is necessary to remove any unreacted labeled standard, before making the determination on the antibody complex, although theoretically, the determination could be made on the removed unreacted portion of the standard.
2. Prior Art
Spector U.S. Pat. No. 3,766,162 discloses a radioimmunoassay for barbituric acids using antibodies generated by synthetic antigens, the subject of the patent. The antigen comprises a barbituric acid hapten coupled to a protein carrier. The barbituric acid has a 5-substituent and is coupled to the carrier by a peptide bond to that substituent. Spector reports, col. 5, lines 41-43, that the antibody will not differentiate between barbituric acids having different substituents in the 5 position.
Spector mentions phenyl as an example of aryl groups among the list of possible derivatives at the 5 position (col. 3, line 5) and states that the bartituric acid derivative may be disubstituted.
The listing of 5-position derivatives in column 3 of Spector also includes mention of other aromatic groups, namely (lines 13-14) carboxy-substituted aralkyl groups e.g. p-carboxy-benzyl, p-carboxy-.alpha.-methyl-benzyl. It will be noted that these groups have an alkyl group connecting the phenyl ring to the barbituric acid ring.
It is clear that the point of coupling is to a carboxy or amino group on a 5 substituent (col. 2 lines 68-71) and many of the possible derivatives listed in column 3 carry carboxyl groups. The Spector patent (col. 3, lines 14-19) states that these carboxyls can be converted to amines.
The general teaching of Spector is that antibodies produced from these antigens will bind with barbituric acids that are mono or di-substituted in the 5 position (col. 2 lines 33-36; col 4 lines 45-57; col. 5 lines 12-15 and 36-55; and col. 7 lines 3-10). The antibodies generally bind with barbituric acids substituted in the 5-position while being able to discriminate some variations elsewhere in the molecule, notably substitution at the 1 or 3 position and a 2-thio derivative (col. 5 lines 36-55 and Col. 7 lines 3-10). It appears that antibodies produced from any one antigen can be expected to bind with any 5-substituted 1, 3-unsubstituted barbituric acid. The particular character of the 5-substituent does not appear to be important. Thus it may be selected from the long list of derivative groups recited at the head of column 3. Further, these derivatives can be extended by reaction of a carboxyl derivative with a diamine, col. 3 lines 19-23.
Column 4, lines 54-57, states inter alia that the antibody will selectively complex with "the substituted barbituric acid". Presumably this is "The barbituric acid derivative" described in the paragraph at column 2, line 64, which according to lines 68-71 must include on the 5-substituent a carboxyl or amino group. There is however no report of the complexing of antibody with such a carboxyl or amino containing barbituric acid.
This invention differs from the Spector patent in that coupling to the carrier is through the meta or para position on the phenyl ring of the hapten compound. The resulting antigen raises an antibody which is specific to the respective hapten compound used. The Spector coupling is to a carboxyl or amino group in the 5 position and the resulting antibodies are not specific to either of the three barbituric acid target haptens, such as phenobarbital, included in this invention.
In fact, as Spector states (col. 5, lines 41-43): "the antibody will not differentiate between barbituric acids having different substituents in the 5-position."